The purpose of this project is to develop vaccines against otitis media caused by nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis. In this fiscal year, efforts are focusing on investigating if intranasal immunization with a detoxified lipooligosaccharide (LOS)-tetanus toxoid (dLOS-TT) conjugate vaccine would generate protective immunity against NTHi in a mouse model of nasopharyngeal colonization. The results demonstrated that intranasal immunization with dLOS-TT plus adjuvant cholera toxin (CT) significantly induced LOS-specific IgA antibodies in mouse external secretions, especially in nasal wash (90-fold), bronchoalveolar lavage fluid (25-fold), saliva (13-fold) and fecal extract (3-fold). LOS-specific IgA antibody forming cells were also found in mucosal and lymphoid tissues with their highest numbers in the nasal passage (528 per 106 cells). In addition, the intranasal immunization elicited a significant rise of LOS-specific IgG (32-fold) and IgA (13-fold) in serum. For the immunized mice which had been challenged through the nose with 107 live NTHi strain 9274, the vaccine group showed a significant reduction (74%-76%) of NTHi, compared to that of control groups with CT alone or dLOS plus CT (p<0.05). Negative correlations were found between bacterial counts and the levels of nasal wash IgA or IgG, saliva IgA or serum IgG. The clearance of five heterologous strains was investigated and revealed a significant clearance in strains 3198, 5657 and 7502 but not in strains 1479 and 2019. These data suggest that intranasal immunization with dLOS-TT vaccine elicits both mucosal and systemic immunity against NTHi colonization in a mouse model of nasopharyngeal colonization. Such a vaccine and vaccination regime may be effective in humans to inhibit NTHi colonization to prevent further NTHi infections.